This research was supported by the National Institute of Mental Health MH 086960-01A1 (Christa K. McIntyre).

All procedures described in this protocol are carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and they were approved by the Institutional Animal Care and Use Committee of The University of Texas at Dallas.
1. Construction of VNS Cuffs
<ol>
	<li>Create a drilling tool by sawing off the sharp end of a 22 &#189; G needle.</li>
	<li>Run the now blunt end of the 22 &#189; G needle over a metal file several times to flatten it. Hold the needle at a 45&#186; angle to the file and run it several times over the file while rotating it. This will cause the metal to become thinner and furl inward. CAUTION: Insert the tip of the scalpel into the cut end of the needle and rotate with some downward force to unfurl the metal.</li>
	<li>Use magnifying binoculars for the remaining steps in section 1.</li>
	<li>Using a scalpel (10 or 15 blade) cut 4 mm segments of tubing.</li>
	<li>Place one 4 mm segment over a small drill bit or other similarly shaped tool. This is to hold the tubing in place while it is being manipulated.</li>
	<li>Drill 4 holes into the tubing (Figure 1A). Holes should make the four points of a 2 mm by 2 mm square and should be clean with no rough edges. 
	NOTE: The drilling tool needs to be resharpened (step 1.2) every 2 or 3 holes. Difficulty with this step is almost certainly due to a drilling tool that is not sharp enough.</li>
	<li>With the tubing still on the drill bit, use a scalpel to cut the tubing lengthwise between the holes so that two holes end up on either side of the cut (Figure 1B).</li>
	<li>Using a sewing needle and suture thread, pass suture through the holes to create the rigging for the ultimate placement of the cuff around the vagus nerve. Start with the needle inside the cuff and pass it through one of the holes, then go back through the adjacent hole from the outside (Figure 1C). Tie the thread together ~2 cm from the plastic so that the tubing and thread make a triangle. Allow ~8 cm of thread after the knot and trim. Repeat the process for the holes on the opposite side of the cut. The tubing is now ready to be wired.</li>
	<li>Prepare wires for cuffs.
	<ol>
		<li>Cut platinum iridium wire into 70 mm segments.</li>
		<li>Using the finest tip for the jewelry torch, create a sharp flame with blue center that is as refined as possible and use it to strip ~1 cm of the plastic coating from the wire. Apply the blue center of the flame to stripped end of the wire to create a small ball. Apply the blue center of the flame to several points of the stripped portion to fuse the seven strands together. The wire in these spots will appear to kink.</li>
		<li>On the opposite end of the wire, apply the blue center of the flame to the end to create a small ball. Minimize stripping the plastic on this end.</li>
	</ol>
	</li>
	<li>Wire the cuff (Figure 1D). 
	NOTE: Steps in section 1.10 must be done under magnifying binoculars.
	<ol>
		<li>Tape the cuff down using the threads so that it is oriented with the cut running horizontally. Pull the threads tight so that the cuff is pulled open and then tape down the threads.</li>
		<li>Grasp the balled end of the stripped side of the prepared wire with #5 forceps and push through the bottom right hole. Pull the forceps out of the hole, leaving the end of the wire in the middle of the cuff.</li>
		<li>Re-grasp the balled end of the stripped wire (now in the middle of the cuff) and push it through the top right hole. Pull the forceps out of the hole, leaving the wire passed completely through the cuff and loose on the top side of the cuff.</li>
		<li>Re-grasp the balled end of the stripped wire (now outside the cuff on the top side) and push it back through the top right hole from the inside. Continue doing so until the wire is securely in place: test by tugging on the opposite end of the wire. 
		NOTE: During this process it is critical to differentiate the stripped/insulated portions of the wire. The wire that is ultimately positioned in the &#8216;trough&#8217; of the cuff must be stripped, but everything below the bottom holes (outside the cuff on the bottom end) must be insulated. This ensures delivery of current only to the vagus nerve.&#160;</li>
		<li>Grasp the balled end of the insulated side and push it through the bottom right hole from the inside of the cuff, looping it around once.</li>
		<li>Repeat steps 1.10.2 &#8211; 1.10.5 on the left side of the cuff so that two wires end up fixed to the tubing, one on the right side and one on the left side.</li>
		<li>Place a gold pin into the arm of the helping hand with the hole facing up. Fill the hole with flux.</li>
		<li>Solder the insulated end of the wire (now attached to the cuff) into the pin.</li>
		<li>Allow the solder to cool, and then melt it again. This ensures a good connection between the end of the wire and the inside of the pin. Apply more solder if necessary. Repeat for second wire.</li>
		<li>With the leads running to the right, mark the top of the cuff with permanent marker. Also mark the gold pin attached to the top lead.</li>
	</ol>
	</li>
</ol>
2. Construction of Headcap for VNS Input Site
<ol>
	<li>Cut 30 mm segments of 26 AWG copper wire. Strip a small portion on each end.</li>
	<li>Cut the narrow end off loose gold pins and solder the stripped end of the wire to the cut end of the gold pin. Create two wire/pin compounds for each desired input site. Place a connector into the helping hands and solder the wire end of a wire/pin compound to the each of the two fluxed teeth of the connector (Figure 2G).</li>
	<li>Mark one of the wires with sharpie. During the surgery, position the implant with the marked wire rostral to the unmarked wire.</li>
</ol>
3. VNS Surgery
<ol>
	<li>Create custom glass tools for handling of vagus nerve during surgery.
	<ol>
		<li>Use borosilicate glass to pull a micropipette so that it has a long tapered tip. If no pipette puller is available, break the glass to create a longer edge.</li>
		<li>Hold the non-tapered end of the pipette with a thick cloth (to prevent burns) and press the tapered/broken end into a smooth fire-resistant surface while applying the blue flame from the jewelry torch to the tapered end. The glass will bend as it is pressed into the surface. Apply flame until a hook or J shape forms.</li>
	</ol>
	</li>
	<li>VNS surgical procedures
	<ol>
		<li>Gather and sterilize all tools. Prepare a sanitary, heated surgical area.</li>
		<li>Anesthetize animal with ketamine/xylazine (85 mg/kg, 5mg/kg, IP). Assess the depth of the anesthetic plane by monitoring the animal&#8217;s vocalizations and withdrawal-reflexes in response to toe and/or tail pinching.</li>
		<li>Shave the top of head and left side of animal&#8217;s neck. Protect the animal&#8217;s eyes with mineral oil or eye ointment. Apply iodine cleansing solution with gauze and then alcohol with gauze to the shaved areas. Repeat once.</li>
		<li>Inject 0.05 ml marcaine subcutaneously on the top of head and allow the bolus to disperse while placing the animal into the stereotaxic instrument.</li>
		<li>Use a scalpel to make an incision in the skin on the skull to expose both lambda and bregma. Prepare a path for the cuff by using blunt forceps to tunnel subcutaneously from the incision site down the left side in front of the ear to the left side of the neck.</li>
		<li>Pull the incision site open with hemostats. Using cotton swabs, apply hydrogen peroxide to the exposed skull to remove any remaining tissue.</li>
		<li>Using a scalpel, drill two shallow starter holes in the skull to place anchor screws. They should be placed far enough apart to allow room for the implant, but not too close to the surrounding tissue. Avoid placing screws directly on the midline.&#160;
		<ol>
			<li>With forceps and screwdriver, drive a bone screw into both holes. The screws should be tight in the holes, with the caps 2 - 3 mm above the surface of the skull to allow room for acrylic to fill in under and around the screws.</li>
		</ol>
		</li>
		<li>Fill the space under, around, and between the screws with a small amount of acrylic, avoiding the surrounding tissue. Then place a larger amount of acrylic in the middle of the skull between the two screws.</li>
		<li>Grab the implant so the marked wire is oriented rostral to the unmarked wire and quickly place the implant in the acrylic, taking care not to get acrylic into the gold pins, the clasp area, or the input site on top. Once positioned allow to set ~5 min until dry. This can also be done using the arm of the stereotaxic for support. Fill in any cracks or gaps between the implant and skull with a low viscosity blend of acrylic. Allow to dry.</li>
		<li>Use magnifying binoculars for the remaining steps in section 3.2.</li>
		<li>Remove the animal from the stereotaxic instrument. Place the animal on its right side, rotating it slightly toward the ventral position.</li>
		<li>Make a small incision approximately over the left jugular vein. The jaw bone and clavicle should be roughly equidistant to the incision site. Widen the incision using blunt dissection until the muscle layer is reached. The sternomastoid, sternohyoid, and omohyoid muscles should be visible. Use muscle retractors to keep the site open.</li>
		<li>Continue blunt dissecting along the natural furrows between the muscles. Look for the pulsing of the carotid artery. Heading through the muscle toward the pulsing will reveal the carotid artery. Pull the muscles back with the muscle retractor. The sheath containing the carotid artery also contains the vagus nerve. Carefully blunt dissect the sheath with the scissors.</li>
		<li>Identify the vagus nerve. It is the largest nerve in the carotid sheath and is usually to the animal&#8217;s left side of the artery but can be found on any side. Switch to the custom glass tools and separate the vagus nerve from the carotid artery. The nerve should be free of any other tissue for at least 5 mm.</li>
		<li>From the incision on the head, using the threads on the side of the cuff opposite the leads, pull the cuff through the previously made subcutaneous tunnel with forceps or small hemostats. Push it through the tissue into the incision site on the neck.</li>
		<li>Gently lift the nerve up using the glass tools and push the threads on the side of the cuff opposite the leads under the nerve. Pull the threads all the way through, taking care not to rub against the nerve. The cuff should be immediately adjacent to the nerve.</li>
		<li>Make sure the cuff is oriented with the marked &#8216;top&#8217; side superior. Drop the nerve into the middle of the cuff. The nerve should now lie across both wires in the trough of the cuff (Figure 2B). Tie the threads together to close the cuff.</li>
		<li>On the headcap, plug the pins attached to the cuff into the gold pins on the stimulation input site. Plug the marked pin into the gold pin attached to the most anterior tooth on the simulation input site.</li>
		<li>To verify that the cuff properly stimulates the vagus nerve, perform a cessation of breathing test by, connecting the stimulator to the stimulation input site on the headcap and running stimulation (0.2 mA, 60 Hz, up to 10 sec). Breathing should briefly stop and heart rate should drop, verifying cuff function.</li>
		<li>Secure the pins on the stimulation input site with acrylic. Cover the wires and verify that exposed pins and wires do not lead to short circuits. Use acrylic to smooth over any bumps or to fill in any gaps. Allow to dry.</li>
		<li>Suture closed both incision sites. Inject 0.05 ml marcaine subcutaneously near the neck incision site. Apply antibiotic ointment to incision sites. Optionally, leave a male connector in place on the stimulation input site to prevent damage or obstruction during the healing process.</li>
		<li>Treat with antibiotic and follow standard post-operative care including appropriate analgesia. Return animals to animal housing facility after they regain mobility. Allow 5 days for recovery. To ensure maximal longevity and function of the headcap, house animals singly for the remainder of the experiment. 
		NOTE: Sham-VNS rats undergo the same surgery, however the circuit is designed to short at the level of the headcap (i.e., a headcap is implanted and the vagus nerve is separated from the carotid artery, but no electrode cuff is placed around the nerve).</li>
	</ol>
	</li>
</ol>
4. Auditory Fear Conditioning
NOTE: This fear conditioning protocol is more intensive than most21 because the goal of these experiments is to enhance extinction. With mild fear conditioning that is easily extinguished, a floor effect can obscure this enhancement.
<ol>
	<li>House animals on a 12 hr&#160;light/dark cycle with ad libitum access to food and water. Handle animals daily during recovery from surgery.</li>
	<li>Set up the conditioning and testing apparatus, consisting of an operant box housed in a sound-attenuated chamber (Figure 2C). The operant box has clear plastic walls, 20 x 20 x 20 cm, and has a stainless steel grid floor which is connected to a foot shock generator. Use a white house-light to illuminate the chamber for video recording. Use a 9 kHz, 85 dB SPL tone as the conditioned stimulus.</li>
	<li>Record behavior using a digital camera located inside the chamber, above the operant box. View and monitor the session on a computer located outside the behavior room. Save videos for later analysis.</li>
	<li>Wipe chambers with 70% ethanol before and after each session to eliminate olfactory cues.</li>
	<li>Fear-condition the rats for 2 days (Figure 3A). Confirm that the rats are not innately afraid of the tone by presenting 5 tones (9 khz, 85 db, 30 sec) on the first day. Ensure that the freezing levels are negligible.
	<ol>
		<li>Follow the initial tone presentations with 8 tone-footshock (1 sec, 0.5 mA) pairings on each of 2 consecutive days. Repeat the tone-footshock pairings again on the second day. Vary the inter-stimulus-interval (ISI) between 2 and 4 min, averaging 3 min for every trial. Randomize the point at which the shock occurs during the tone.</li>
	</ol>
	</li>
	<li>On the third day, test the strength of the tone/shock association.&#160;Play 4 tones with an ISI of 3, 4, or 5 min (4 min average) in the absence of footshocks and record the animals&#8217; freezing behavior during the tone presentations and during the inter-stimulus-intervals as measures of the conditioned fear response (CFR).</li>
	<li>On day 4, begin extinction training with VNS or Sham VNS.
	<ol>
		<li>Plug the rats into the stimulator by inserting the male connectors from the stimulator into the stimulation input site. Place animals into the chamber (Figure 2A, 2C). Set the stimulator to 0.4 mA, 500 &#181;s pulse width at 30 Hz. Set stimulation to a total duration of 30.15 sec, starting 150 msec before the onset of the tone. Play animals 4 tones (as in step 4.6) and pair each tone presentation with VNS or Sham VNS.</li>
	</ol>
	</li>
	<li>Periodically test the electrical integrity of cuff and input site using an oscilloscope.
	<ol>
		<li>Plug the animal in as normal and split the output from the stimulator to the oscilloscope.</li>
		<li>Set the range on the oscilloscope as -20 V to +20 V and run stimulation. The waveform of the 30 Hz stimulation should be visible on the oscilloscope. Stimulations exceeding 10 V in size indicate high impedance and an improperly functioning cuff or connection at the stimulation input site.</li>
	</ol>
	</li>
	<li>To test the effect of VNS on extinction training run a second CFR test (as in step 4.6) on day 5. Record the time spent freezing during tone presentations and compare to baseline freezing recorded during the first CFR test (step 4.6).</li>
	<li>Analyze the videos using an independent observer who is blind to the treatment conditions. Measure time spent freezing during tone presentations using a stopwatch. Freezing is defined as complete immobility, during which the rat exhibits rapid respiration, lowered head, and spread paws22. Analysis of freezing behavior can be split into two phases: during tone presentation and during the inter-stimulus interval.&#160;</li>
</ol>
5. In Vivo Recordings of Evoked Field Potentials
Note: This step is optional. Evoked field potentials (EFPs) are recorded 24 hr&#160;after tests of reinstatement (Day 5) in isoflurane-anesthetized rats mounted in a stereotaxic apparatus, following standard procedures23,24.
<ol>
	<li>Gather and sterilize all tools.</li>
	<li>Induce anesthesia with isoflurane (5% in 100% oxygen, flowrate 1l/min) in a clear plastic chamber. Assess the depth of the anesthetic plane by monitoring the animal&#8217;s vocalizations and withdrawal-reflexes in response to toe and/or tail pinching. Use mineral oil or eye ointment to protect eyes.</li>
	<li>Inject 0.05 ml marcaine subcutaneously on the top of head and allow the bolus to disperse. Use a scalpel and hemostats to remove the headcap from the skull. Use as little force as possible to avoid obscuring bregma.</li>
	<li>Place the animal in the stereotaxic instrument. Use a scalpel to widen the incision to expose both lambda and bregma. Maintain anesthetic plane with isoflurane (3% in 100% oxygen, flowrate 1 L/min) via a nosecone.</li>
	<li>Drill holes into the skull above the infralimbic prefrontal cortex (IL) and the basolateral amygdala (BLA). Lower a glass microelectrodes (2M KCl; 1-2 MOhms resistance) into the BLA (D/V: 7.2, A/P: 2.7, M/L: 4.9 from bregma) and a stimulation electrode into the IL region of the medial prefrontal cortex (D/V: 4.6, A/P: 3.0, M/L: 0.7 from bregma) (Figure 4A).</li>
	<li>Stimulate the IL to evoke EFPs in the BLA. Data shown in Figure 4 was acquired with the following settings: A stimulation pulse of 0.3 msec duration, using a stimulation intensity that corresponded to 40% of the minimum current intensity that evoked a maximum field response (based on an input&#8211;output curve determined before collection of baseline data), delivered every 15 sec.</li>
	<li>Collect baseline data for a minimum of 10 - 15 min before inducing synaptic plasticity. 
	NOTE: The protocol used to evoke synaptic plasticity will vary with the requirements of the experiments and must be carefully selected by each experimenter. Data in Figure 4C shows changes in the EFP following 3 bursts of 100 pulses at 50 Hz (2 sec), with 20 sec inter-burst intervals at the minimum current intensity that evoked the maximum field response.</li>
	<li>Measure the amplitude of the EFP as the difference between the mean of a 5 msec window before the stimulation artifact and the mean of a 5 msec window around 20 - 25 msec after the stimulation artifact, corresponding to the negative peak of the field potential. Normalize data to baseline and set the average of a 10 min baseline as 100%. Use the averaged EFP amplitudes of another 10 min period after plasticity induction (e.g., 40 - 50 min post induction) to assess long-term changes in EFP amplitude.</li>
	<li>After the end of the recording decapitate the anesthetized animal and extract the brain. Prepare tissue for histological verification of electrode placement.</li>
</ol>

The authors have no competing interests or conflicts.

We present here a protocol that is used to facilitate extinction of conditioned fear during a single session of exposure to conditioned cues19 and to modulate plasticity in the pathway between the infralimbic cortex and the basolateral amygdala that may mediate extinction learning20. A crucial step for the success of this protocol is the proper delivery of VNS during extinction training. Therefore, special care should be given to the construction of the cuff electrodes and the placement of the cuff ...

Classical fear conditioning provides a widely used animal model to study the biological basis of anxiety disorders. During fear conditioning, an aversive stimulus (the unconditioned stimulus, US, e.g., a footshock) is presented in conjunction with a neutral stimulus, such as a tone and/or a context (the conditioned stimulus; CS). During fear conditioning, associations between the CS and the US are formed. Eventually the presentation of the CS alone elicits a fear response (the conditioned response; CR). In fear extinction, the CS is presented repeatedly in the absence of the US, causing the CR to gradually diminish1. Thus, extinction of conditioned fear is an active process in which fearful behavioral responses to neutral stimuli are attenuated when they no longer predict aversive outcomes. Extinction of conditioned responses requires consolidation of new memories that compete with learned associations. A hallmark of anxiety disorders is impaired extinction2-4. Thus, extinction of conditioned fear in animal models serves as an important paradigm both for inhibitory learning and as a model of behavior therapy for human anxiety disorders5,6.
Because there is close correspondence between fear and human anxiety, it is thought that these studies can provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder and will help to develop strategies to treat them. An important goal of preclinical research is to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate extinction learning. Vagus nerve stimulation (VNS) is a minimally invasive neuroprosthetic approach that might be used to provide tight temporal and circuit-specific modulation of brain areas and synapses engaged in an ongoing task. A series of recent studies from Michael Kilgard&#39;s group at The University of Texas at Dallas have shown that pairing VNS with discrete sensory or motor stimuli (e.g., a tone or a lever pull) is highly effective in promoting cortical plasticity to treat tinnitus7, or to overcome motor deficits following stroke8-10. In addition, non-contingent VNS that occurs within a short time-window after learning similarly promotes cortical plasticity and enhances memory consolidation in rats and in humans11-13.
Considering the role of the vagus nerve in the parasympathetic pathway, it is not surprising that it could participate in modulating memories and synaptic plasticity. Highly emotional events tend to produce stronger memories than non-emotional memories. This is likely due to the influence of stress hormones on memory consolidation. Posttraining administration of the stress hormone adrenaline enhances memory consolidation in human and non-human animals, but adrenaline does not cross the blood-brain-barrier14, 15. Therefore, stress-induced adrenaline release must impact the brain indirectly to enhance memory consolidation. Strong evidence suggests that the vagus nerve may be the link between circulating adrenaline and the brain. Miyashita and Williams16 found that systemic administration of adrenaline increased vagal nerve firing, and increased levels of norepinephrine in the amygdala17. Systemic administration of adrenaline does not enhance memory consolidation when &#946;-adrenergic receptors are blocked in the amygdala18 suggesting that the vagus nerve plays a role in the pathway that turns emotionally arousing experiences into long-term memories.
Thus, pairing VNS with training has the potential to enhance the brain changes that support memory consolidation and exposure to conditioned cues in the absence of reinforcement enhances the consolidation of extinction learning in rats19,20. Here we describe the use of VNS as a tool to promote cortical plasticity and facilitate extinction of a conditioned fear response.

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			<td>AmScope</td>
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			<td>A-M Systems&#160;</td>
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			<td>Walgreens</td>
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		</tr>
		<tr>
			<td>Tubing</td>
			<td>Braintree Scientific Inc</td>
			<td>MRE-065</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum Iridium Wire</td>
			<td>Medwire</td>
			<td>10IR9/49T</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold Pins</td>
			<td>Mill-Max</td>
			<td>1001-0-15-15-30-27-04-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture Thread</td>
			<td>Henry Schein</td>
			<td>100-5797</td>
			<td></td>
		</tr>
		<tr>
			<td>22 G Needles</td>
			<td>Fisher&#160;</td>
			<td>14-815-525</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper Tape</td>
			<td>Fisher&#160;</td>
			<td>03-411-602</td>
			<td></td>
		</tr>
		<tr>
			<td>Solder</td>
			<td>Home Deopt</td>
			<td>327793</td>
			<td></td>
		</tr>
		<tr>
			<td>Flux&#160;</td>
			<td>Home Deopt</td>
			<td>300142</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Blade, 10 or 15</td>
			<td>Stoelting</td>
			<td>52173-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Silastic Laboratory Tubing .51 mm ID x .94 mm OD</td>
			<td>Fisher&#160;</td>
			<td>508-002</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Headcaps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Connector Pieces (male)</td>
			<td>Omnetics Connector Corporation</td>
			<td>A25001-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Headcap pieces (female)</td>
			<td>Omnetics Connector Corporation</td>
			<td>A24001-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Teets Dental Acrylic, Liquid and Powder</td>
			<td>A-M Systems</td>
			<td>525000, 526000</td>
			<td></td>
		</tr>
		<tr>
			<td>26 Gauge Solid Copper Wire</td>
			<td>Staples</td>
			<td>1016882 &#160;</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgery</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bone Screws</td>
			<td>Stoelting+CB33:C61</td>
			<td>51457</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Blades, 10 or 15</td>
			<td>Stoelting</td>
			<td>52173-10</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringes</td>
			<td>Fisher</td>
			<td>14-826-261</td>
			<td></td>
		</tr>
		<tr>
			<td>22 G Needles</td>
			<td>Fisher&#160;</td>
			<td>14-815-525</td>
			<td></td>
		</tr>
		<tr>
			<td>27 G Needles</td>
			<td>Fisher</td>
			<td>14-826-48</td>
			<td></td>
		</tr>
		<tr>
			<td>2&#34; x 2&#34; Gauze</td>
			<td>Fisher</td>
			<td>22-362-178</td>
			<td></td>
		</tr>
		<tr>
			<td>Swabs</td>
			<td>Fisher</td>
			<td>19-120-472</td>
			<td></td>
		</tr>
		<tr>
			<td>Puppy Pads</td>
			<td>PetCo</td>
			<td>1310747</td>
			<td></td>
		</tr>
		<tr>
			<td>Kim Wipes</td>
			<td>Fisher</td>
			<td>06-666-A</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Chamber and Behavioral Setting&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Husky Metal Front Base Cabinet (30WX19DX34H)</td>
			<td>Home Depot</td>
			<td>100607961</td>
			<td></td>
		</tr>
		<tr>
			<td>Quiet Barrier&#173; HD Soundproofing Material (Sheet) (PSA)</td>
			<td>soundproofcow.com</td>
			<td>10203041</td>
			<td></td>
		</tr>
		<tr>
			<td>Convoluted Acoustic Foam Panel</td>
			<td>soundproofcow.com</td>
			<td>10432400</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolated Pulse Stimulator Model 2100</td>
			<td>A-M Systems</td>
			<td>720000</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Camera - Logitech Webcam C210</td>
			<td>Logitech</td>
			<td>B003LVZO88</td>
			<td></td>
		</tr>
		<tr>
			<td>MatLab</td>
			<td>Mathworks.com</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sinometer 10MHz Single Channel Oscilloscope</td>
			<td>Sinometer</td>
			<td>CQ5010C</td>
			<td></td>
		</tr>
		<tr>
			<td>OxyLED T-01 DIY Stick-on Anywhere 4-LED Touch Tap Light</td>
			<td>OXYLED</td>
			<td>B00GD8OKY0</td>
			<td></td>
		</tr>
		<tr>
			<td>5k ohm potentiomter</td>
			<td>Alpha Electronics</td>
			<td>B00CTWDHIO</td>
			<td></td>
		</tr>
		<tr>
			<td>Extech 407730 40-to-130-Decibel Digital Sound Level Meter</td>
			<td>Extech Instruments</td>
			<td>B000EWY67W</td>
			<td></td>
		</tr>
		<tr>
			<td>DSCK-C Dual Output, scrambled shocker</td>
			<td>Kinder Scientific Co</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

vagus nerve stimulation as a tool to induce plasticity in pathways relevant for extinction learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

This section illustrates examples of results that can be obtained by using VNS in combination with extinction learning to reduce the expression of the conditioned fear response in rats. For Days 1 and 2 (Auditory Fear Conditioning), rats were trained on an auditory fear conditioning task in which footshocks were paired with a tone. On Day 3 (Pre Treatment Test), tones were presented in the absence of footshocks to measure freezing levels and infer conditioned fear response acquisition. On Day 4 (Treatment) rats received ...

Watch this Scientific Journal Video about Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning at JoVE.com

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

vagus-nerve-stimulation-as-tool-to-induce-plasticity-pathways

Research

JoVE Journal

Behavior

23.1K Views.  The University of Texas at Dallas. Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

Video: Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

The Crossmodal Congruency Task as a Means to Obtain an Objective Behavioral Measure in the Rubber Hand Illusion Paradigm

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden hand is touched. If the viewed and felt touches are given at the same time then this is sufficient to induce the compelling experience that the rubber hand is one's own hand. The RHI can be used to investigate exactly how the brain constructs distinct body representations for one's own body. Such representations are crucial for successful interactions with the external world. To obtain a subjective measure of the RHI, researchers typically ask participants to rate statements such as "I felt as if the rubber hand were my hand". Here we demonstrate how the crossmodal congruency task can be used to obtain an objective behavioral measure within this paradigm.
The variant of the crossmodal congruency task we employ involves the presentation of tactile targets and visual distractors. Targets and distractors are spatially congruent (i.e. same finger) on some trials and incongruent (i.e. different finger) on others. The difference in performance between incongruent and congruent trials - the crossmodal congruency effect (CCE) - indexes multisensory interactions. Importantly, the CCE is modulated both by viewing a hand as well as the synchrony of viewed and felt touch which are both crucial factors for the RHI.
The use of the crossmodal congruency task within the RHI paradigm has several advantages. It is a simple behavioral measure which can be repeated many times and which can be obtained during the illusion while participants view the artificial hand. Furthermore, this measure is not susceptible to observer and experimenter biases. The combination of the RHI paradigm with the crossmodal congruency task allows in particular for the investigation of multisensory processes which are critical for modulations of body representations as in the RHI.

We demonstrate how an objective measure can be employed in the widely employed rubber hand illusion paradigm. This measure is obtained by modifying the well-established crossmodal congruency task. This task allows the investigation of multisensory processes which are critical for modulations of body representations as in the rubber hand illusion.

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden ...

Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that neural mechanisms for certain behavioral tasks and response behaviors are different in females and males. Using females in research studies can represent a challenge because of the variation of gonadal hormones during their estrous cycle. This protocol describes well-established procedures that are useful in investigating the role of estrogen in fear extinction memory consolidation in female rats. Phase of the estrous cycle and exogenous estrogen administration prior to extinction training can influence extinction recall 24 hr later. The vaginal swabbing technique for estrous phase identification described here aids the examination and manipulation of naturally cycling gonadal hormones. The use of this basic rodent model may further delineate the mechanisms by which estrogen can modulate fear extinction memory in females.

Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that ...

Quantifying Learning in Young Infants: Tracking Leg Actions During a Discovery-learning Task

Task-specific actions emerge from spontaneous movement during infancy. It has been proposed that task-specific actions emerge through a discovery-learning process. Here a method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process. This discovery-learning task uses an infant activated mobile that rotates and plays music based on specified leg action of infants. Supine infants activate the mobile by moving their feet vertically across a virtual threshold. This paradigm is unique in that as infants independently discover that their leg actions activate the mobile, the infants&#8217; leg movements are tracked using a motion capture system allowing for the quantification of the learning process. Specifically, learning is quantified in terms of the duration of mobile activation, the position variance of the end effectors (feet) that activate the mobile, changes in hip-knee coordination patterns, and changes in hip and knee muscle torque. This information describes infant exploration and exploitation at the interplay of person and environmental constraints that support task-specific action. Subsequent research using this method can investigate how specific impairments of different populations of infants at risk for movement disorders influence the discovery-learning process for task-specific action.

A method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process.

Task-specific actions emerge from spontaneous movement during infancy. It has been proposed that task-specific actions emerge through a discovery-learning ...

A Treatment Package without Escape Extinction to Address Food Selectivity

Feeding difficulties and feeding disorders are a commonly occurring problem for young children, particularly children with developmental delays including autism. Behavior analytic interventions for the treatment of feeding difficulties oftentimes include escape extinction as a primary component of treatment. The use of escape extinction, while effective, may be problematic as it is also associated with the emergence of challenging behavior (e.g., extinction burst). Such challenging behavior may be an acceptable side effect in treatment cases where feeding problems are severe and chronic (e.g., failure to thrive). However, in more acute cases (e.g., selective eating), the negative side effect may be unwarranted and undesired. More recent research on the behavioral treatment of food selectivity has begun to evaluate treatments for feeding difficulties that do not include escape extinction (e.g., demand fading, behavioral momentum), with some success. However, research to date reveals individual differences in responsiveness to such treatments and no clear preferable treatment has emerged. This manuscript describes a multi-component treatment package that includes shaping, sequential presentation and simultaneous presentation, for the treatment of food selectivity in four young children with developmental delays. This treatment package extends the literature on the behavioral treatment for food selectivity and offers a multi-component treatment protocol that may be clinically applicable across a range of treatment scenarios and settings.

Feeding difficulties are a common problem for children with developmental disorders, including autism, and behavioral interventions often include escape extinction. Recent research has begun to evaluate treatments that do not include escape extinction. This manuscript describes a multicomponent treatment package that does not use escape extinction to treat feeding difficulties.

Feeding difficulties and feeding disorders are a commonly occurring problem for young children, particularly children with developmental delays including ...

Combined Shuttle-Box Training with Electrophysiological Cortex Recording and Stimulation as a Tool to Study Perception and Learning

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the necessary equipment is now available by several commercial companies. This protocol provides a detailed description of a two-way shuttle-box avoidance learning paradigm in rodents (here Mongolian gerbils; Meriones unguiculatus) in combination with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical electrophysiological in vivo recordings. The detailed protocol is applicable to study multiple aspects of learning behavior and perception in different rodent species.
Site-specific ICMS of auditory cortical circuits as conditioned stimuli here is used as a tool to test the perceptual relevance of specific afferent, efferent and intracortical connections. Distinct activation patterns can be evoked by using different stimulation electrode arrays for local, layer-dependent ICMS or distant ICMS sites. Utilizing behavioral signal detection analysis it can be determined which stimulation strategy is most effective for eliciting a behaviorally detectable and salient signal. Further, parallel multichannel-recordings using different electrode designs (surface electrodes, depth electrodes, etc.) allow for investigating neuronal observables over the time course of such learning processes. It will be discussed how changes of the behavioral design can increase the cognitive complexity (e.g. detection, discrimination, reversal learning).

Shuttle-box avoidance learning is well-established in behavioral neuroscience. This protocol describes how shuttle-box learning in rodents can be combined with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical in vivo recordings as a tool to study multiple aspects of learning and perception.

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the ...

Using a Virtual Store As a Research Tool to Investigate Consumer In-store Behavior

People&#39;s responses to products and/or choice environments are crucial to understanding in-store consumer behaviors. Currently, there are various approaches (e.g., surveys or laboratory settings) to study in-store behaviors, but the external validity of these is limited by their poor capability to resemble realistic choice environments. In addition, building a real store to meet experimental conditions while controlling for undesirable effects is costly and highly difficult. A virtual store developed by virtual reality techniques potentially transcends these limitations by offering the simulation of a 3D virtual store environment in a realistic, flexible, and cost-efficient way. In particular, a virtual store interactively allows consumers (participants) to experience and interact with objects in a tightly controlled yet realistic setting. This paper presents the key elements of using a desktop virtual store to study in-store consumer behavior. Descriptions of the protocol steps to: 1) build the experimental store, 2) prepare the data management program, 3) run the virtual store experiment, and 4) organize and export data from the data management program are presented. The virtual store enables participants to navigate through the store, choose a product from alternatives, and select or return products. Moreover, consumer-related shopping behaviors (e.g., shopping time, walking speed, and number and type of products examined and bought) can also be collected. The protocol is illustrated with an example of a store layout experiment showing that shelf length and shelf orientation influence shopping- and movement-related behaviors. This demonstrates that the use of a virtual store facilitates the study of consumer responses. The virtual store can be especially helpful when examining factors that are costly or difficult to change in real life (e.g., overall store layout), products that are not presently available in the market, and routinized behaviors in familiar environments.

This paper describes the use of a desktop virtual store to create virtual shopping environments to investigate in-store consumer behavior. A description of the protocol to build and run experiments, example results from an experiment concerning store layout, and important considerations when conducting virtual store experiments are presented.

People&#39;s responses to products and/or choice environments are crucial to understanding in-store consumer behaviors. Currently, there are various ...

Large Volume, Behaviorally-relevant Illumination for Optogenetics in Non-human Primates

This protocol describes a large-volume illuminator, which was developed for optogenetic manipulations in the non-human primate brain. The illuminator is a modified plastic optical fiber with etched tip, such that the light emitting surface area is &#62; 100x that of a conventional fiber. In addition to describing the construction of the large-volume illuminator, this protocol details the quality-control calibration used to ensure even light distribution. Further, this protocol describes techniques for inserting and removing the large volume illuminator. Both superficial and deep structures may be illuminated. This large volume illuminator does not need to be physically coupled to an electrode, and because the illuminator is made of plastic, not glass, it will simply bend in circumstances when traditional optical fibers would shatter. Because this illuminator delivers light over behaviorally-relevant tissue volumes (&#8776; 10 mm3) with no greater penetration damage than a conventional optical fiber, it facilitates behavioral studies using optogenetics in non-human primates.

A protocol to build a tissue penetrating illuminator for delivering light over large volumes with minimal diameter is presented.

This protocol describes a large-volume illuminator, which was developed for optogenetic manipulations in the non-human primate brain. The illuminator is a ...

Applying an eMASS Customization Program as a Research Tool to Evaluate Consumer Benefits

As many scholars and practitioners study personalization and relationship marketing, it is important to provide personalization such as mass customization through marketing technology. The purpose of this study is to examine how to conduct consumer research using an online survey and analysis of data. This study examines consumers&#39;&#160;perceived benefits while customizing a product as well as emotional product attachment, attitudes toward a customization program, and loyalty intentions in the context of online retailing. In addition, this study investigates how consumer responses are different based on individual characteristics such as fashion innovativeness. An online survey company in South Korea recruited 290 female apparel shoppers who purchased apparel online. To enhance external validity, this study used an existing retail website with a well-established mass customization program. After completing the customization program, participants complete the online questionnaire. Structural equation modeling (SEM) and latent mean analyses (LMAs) are then performed for analyses. This study stresses the importance of testing measurement invariance for mean comparisons. Before the SEM and LMA, this study follows the hierarchy of invariance tests (configural invariance test, metric invariance test, and scalar invariance test), which are not considered by traditional approaches such as ANOVA. These statistical analyses provide applicability of the invariance test procedures and LMA to consumer behaviors. The conclusions of mean differences have integrity and validity because they are guided by a sophisticated statistical procedure to ensure measurement invariance.

Presented here is a protocol to examine consumer responses toward mass customization in the context of online retailing. The protocol details the online survey procedure and how to analyze data using structural equation modeling and group differences using latent mean analyses.

As many scholars and practitioners study personalization and relationship marketing, it is important to provide personalization such as mass customization ...

A Methodology for Capturing Joint Visual Attention Using Mobile Eye-Trackers

With the advent of new technological advances, it is possible to study social interactions at a microlevel with unprecedented accuracy. High frequency sensors, such as eye-trackers, electrodermal activity wristbands, EEG bands, and motion sensors provide observations at the millisecond level. This level of precision allows researchers to collect large datasets on social interactions. In this paper, I discuss how multiple eye-trackers can capture a fundamental construct in social interactions, joint visual attention (JVA). JVA has been studied by developmental psychologists to understand how children acquire language, learning scientists to understand how small groups of learners work together, and social scientists to understand interactions in small teams. This paper describes a methodology for capturing JVA in colocated settings using mobile eye-trackers. It presents some empirical results and discusses implications of capturing microobservations to understand social interactions.

Using multimodal sensors is a promising way to understand the role of social interactions in educational settings. This paper describes a methodology for capturing joint visual attention from colocated dyads using mobile eye-trackers.

With the advent of new technological advances, it is possible to study social interactions at a microlevel with unprecedented accuracy. High frequency ...

Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on animals and humans. Adult neurogenesis mechanism is also linked to memory and learning improvement. Traditionally, researchers used to assess learning and memory parameters in rodent models by behavioral tasks. Therefore, the simultaneous monitoring of behavioral changes and EEG is particularly interesting in correlating data between brain activity and task-related behaviors. However, most of the equipment required to perform both studies are either complex, expensive, or uses a wired setup network that hinders the natural animals&#8217; movement. In this study, EEG was recorded with a wireless electrophysiology device along with the execution of a novel object recognition task (NORT). The animal&#8217;s behavior was monitored simultaneously by a video tracking system. Both recordings were analyzed offline by their timestamps which were synched to link EEG signals with the animal&#8217;s actions. Subjects consist of adult Wistar rats after medium-term environmental enrichment treatment. Six skull screw electrodes were fixed in pairs on both hemispheres over frontal, central, and parietal regions and were referenced to an electrode located posterior of the nasal bone. NORT protocol consists of exposing the animal to two identical objects for 10 min. After 2 h and 24 h, one of the objects was replaced with a novel one. Exploration time for each object was monitored by a behavioral tracking software (BTS) and EEG data recording. The analysis of the EEG synced with behavioral data consists of estimations of alpha and beta relative band power and comparisons between novel object recognition versus familiar object exploration, between three experimental stages. In this manuscript, we have discussed electrodes manufacturing process, epidural electrodes implantation surgery, environmental enrichment protocol, NORT protocol, BTS setup, EEG &#8211; BTS coupling for simultaneous monitoring in real-time, and EEG data analysis based on automatic events detection.

The protocol presented here provides information on the simultaneous electroencephalography (EEG) and behavioral assessment in real-time. We have discussed all steps involved in this protocol as an attractive solution for researchers in many fields of neuroscience, particularly in learning and memory areas.

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on ...